Synthesis of N-arylidene-2-(2-Phenoxyphenyl) Acetohydrazides as Anti-Inflammatory Agents.

Diclofenac sodium has been used for its anti-inflammatory actions for about 28 years, but since all the non-steroidal anti-inflammatory drugs (NSAIDs) suffer from the lethal gastro intestinal (GI) toxicities, diclofenac sodium is not an exception. The free -COOH group is thought to be responsible for the GI toxicity associated with all traditional NSAIDs. In the present research, the main motto was to develop new chemical entities as potential anti-inflammatory agents with no GI toxicities. A new type of 2-(2-phenoxyphenyl) acetohydrazide possessing N-arylidene substituents, was synthesized for evaluation as anti-inflammatory agents. The starting material 2-(2-Phenoxyphenyl) acetohydrazide was synthesized from 2-phenoxybenzoic acid in several steps according to the previous published method. Various substituted arylidene-2-phenoxynicotinic acid hydrazide derivatives were synthesized by the reaction of hydrazide 17 with selected aldehydes and screened for their potential anti-inflammatory activity. The structure of synthesized compounds was confirmed by different nuclear magnetic resonance technique, Fourier transform infrared spectroscopy (FTIR) and Mass-spectrometry data format. Qualitative structure-activity relationship data, acquired using the carrageenan-induced rat paw edema assay, showed that this group of arylidene-2-phenoxybenzoic acid hydrazides exhibit anti-inflammatory activity with significant reduction of rat paw edema (17-58% reduction in inflammation at different time intervals) in comparison with control group and a moderate to good activity range in comparison with diclofenac as the reference drug. Compounds 9a, 9d and 9e exhibited the most prominent and consistent anti-inflammatory activity. The compound, N-(4-Chlorobenzylidene)-2-(2-phenoxyphenyl) acetohydrazide (9d), exhibited the most in-vivo activity (32-58% reduction in inflammation) compared to the reference drug diclofenac (35-74% reduction in inflammation) in a carrageenan induced rat paw-edema assay.


Introduction
(NSAIDs) are the most prescribed drugs particularly for different types of arthritis (1-3). The pharmacological activity of NSAIDs inhibition of cyclooxygenase-derived (COX) prostaglandin synthesis is also responsible for the gastro intestinal (GI), renal, and hepatic side effects observed in patients undergoing long term treatment (4-9). It was discovered that cyclooxygenase exists in two isoforms, COX-1 and COX-2, which are regulated and expressed differently. COX-1 provides cytoprotection in the gastrointestinal tract (GIT), whereas inducible COX-2 selectively most of the currently available NSAIDs in the market show greater selectivity for COX-1 compared to COX-2, chronic use of NSAIDs, including diclofenac, may elicit appreciable GI irritation, bleeding and ulceration (12). The GI damage from NSAID is generally attributed to two factors such as local irritation by the direct contact of carboxylic acid (-COOH) moiety of NSAID with GI mucosal cells (topical effect) and decreased tissue prostaglandin production in tissues which undermines the physiological role of cytoprotective prostaglandins in maintaining GI health and homoeostasis (13). Therefore, drugs represents a challenging goal for such a research area. Synthetic approaches based upon and, in turn, the therapeutic window of these NSAIDs.
Diclofenac, Mefenamic acid and Indomethacin are three traditional NSAIDs belonging to the class of fenamates (Figure 1). This class includes derivatives of N-phenyl (or N N heteroaryl) anthranilic acid that are employed pyretic properties. Unlike most of the NSAIDs, the fenamates also appear to compete with prostaglandins for binding at the prostaglandin receptor site and, thus, potentially antagonizing the physiopathological effects of prostaglandins that have already been formed. Fenamates are still endowed with most of the adverse effects induced by NSAIDs, particularly gastrointestinal (GI) bleeding, ulceration, and perforation. Several studies have described the derivatization of the carboxylate function of representative NSAIDs with amide or N-acylarylhydrazone N N having less acidic amide hydrogen resulted in reduced ulcerogenicity (14-17) ( Figure 2). Some evidences suggest that the hydrazone moiety possesses a pharmacophoric character for the inhibition of cyclooxygenase (15). N-Acylhydrazones (NAH) have been widely N N nociceptive, and anti-platelet compounds, due to their ability to mimic the bis-allylic moiety of unsaturated fatty acids and amides, for example, arachidonic acid (AA), precursor of the eicosanoid biosynthesis, involved in the endocannabinoid system. This can be rationalized through the relative acidity in amide hydrogen of NAH group as well as its capacity of stabilizing the free radicals (14). Considering the above results and as a part of our ongoing program to design analgesic and antithe design, synthesis and biological evaluation of a novel diverse group in N-acylarylhydrazone N N derivatives of 2-phenoxybenzoic acid 9a-g with different substituents on the terminal phenyl ring ( Figure 2).

Chemistry
Melting points were determined with a Reichert-Jung hot-stage microscope (Reichert-Jung, Germany) and are uncorrected. Infrared spectra were recorded on a Nicolet Magna 550-FT spectrometer (Nicolet, Madison, WI, USA). 1 H-NMR (500 MHz) spectra were measured through a Brucker FT-500 MHz spectrometer (Bruker Bio Sciences, USA) in CDCl 3 or DMSO-d 6 with tetramethylsilane (TMS) as the internal standard, where j-values (coupling constant) are estimated in Hertz. Spin multiples are given as s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), m (multiplet), and b (broad). Mass spectra were obtained, using a Finnigan Mat triple-stage quadrupole (TSQ 70) spectrometer (Thermo-Electron, Germany). All solvents and reagents were purchased from the Fluka (Aldrich, UK), or Merck Chemical Company (Merck, Germany). Male Wistar rats purchased from Pasteur Institute (Karaj, Iran) and experiments were carried out using protocols approved by the ethics committee of Tehran University of Medical Sciences. Compounds 10-16 have been synthesized according to the published methods in literatures.

N -B e n z y l i d e n e -2 -( 2 -p h e n o x y p h e n y l ) acetohydrazide (9a)
M.p.: 169-171 C (Ethanol/Water); as a mixture of diastereomers in ratio of 2

N -( 4 -M e t h y l -b e n z y l i d e n e ) -2 -( 2phenoxyphenyl) acetohydrazide (9b)
M.

N-(2, 4-Dimethoxybenzylidene)-2-(2phenoxyphenyl) acetohydrazide (9f)
M.p.: 250-252 C (Ethanol/Water); as a mixture of diastereomers in ratio of 1  determined in-vivo using the carrageenaninduced rat paw edema test. Edema was induced in the right hind paw of all rats by subcutaneous (Sigma-Aldrich, Dorset, UK) in saline into their footpads 0.5 h after the intra-peritoneal (IP) administration of compounds (18). The paw thickness was measured from the ventral to the dorsal surfaces using a dial caliper immediately before and then two and four h after the carrageenan injection. The edema was calculated as the thickness variation between thickness of paw before and after carrageenan injection. expressed similar to the inhibition percentage of edema when compared with the control group and was calculated using the following formula: In this formula, T t and T c thickness variation of test group and control group, respectively.
2-(2-Phenoxyphenyl) acetohydrazide (17) is the key intermediate for the production of compound 10 using thionyl chloride in ethanol afforded the ethyl esters 11 in 99% yield (19, 20). Next, hydroxyl compound 12 was prepared, in 95% yield by the reduction of compound 11 with lithium aluminum hydride in Tetrahydrofuran/ Tetrahydrofolic acid/Tetrahydrofolate (THF) (20). The substitution of hydroxyl to chloride compound 13 was obtained in 97% yield by the reaction of compound 12 with thionyl chloride in benzene/pyridine (21). The treatment of compound 13 with sodium cyanide and subsequent hydrolysis of the synthesized compound 14, gave 2-(2-phenoxyphenyl) acetic acid (15) in 87 and 98% respectively. Reaction of 2-(2-phenoxyphenyl) acetic acid (15) with thionyl chloride in ethanol, furnished the ethyl 2-(2-phenoxyphenyl) acetate (16) in high yield after treatment with hydrazine hydrate in absolute methanol, it resulted in the formation of the required hydrazide 17, the key intermediate of this synthesis route, in 94% yield (20,22). Finally, the title hydrazone compounds (9a-g) were prepared in great yields by the condensation of hydrazide 17 with different substituted aromatic aldehydes in ethanol, using hydrochloric acid as catalyst (22)(23)(24)(25) Table 2 are reported in experimental protocols. The FTIR spectra of Schiff bases exhibited very similar features and showed the expected bands for the characteristic groups which were present in the compounds such as C-H and the band for Ar-C-N vibrations. Compounds of 9a-g have C=O stretching bands in the range of 1677-1685. In the proton 1 H-NMR spectral data, all protons were seen according to the expected chemical shift and integral values. The aromatic protons appeared as multiplet peaks within the range of 6.9-7.7 ppm and the singlet signals derived from hydrazide 17 (-NH-NH 2 ) structure appeared at 3.76 ppm. Methylene protons resonated as singlet at 4.1-4.2 ppm. The 1 H-NMR spectra of compounds 9a-g displayed singlet due to -NH-groups around 8.1, 9.01 and 9.4 ppm (probably due to their ability to get exchanged with D 2 O) while each signal showed integration for one proton. For the compounds 9a-g, the signals which belonged to benzylidene group were observed at aromatic region, while the signals of -NHNH 2 disappeared indicating the functionalization of hydrazide to hydrazone with substituted aromatic aldehydes.
The next step of this work was to determine bond of arylidene benzoic acid derivatives 9a-g, in order to assure the stereoisomeric ratio essential for the complete understanding of the biological effect. The careful analysis of the 1 H-NMR for 9a-g, enabled us to detect the presence of two singlet signals related to the imine hydrogen, which was attributed to respective (E ( ( ) and ( E )diastereomers. The existent ratio between the two integration of imine-attached hydrogen in the corresponding 1 H-NMR spectra (Table 1).
In-vivo pharmacological evaluation of 9a-g was carried out to assess their potential antiactivity relationship data acquired using the carrageenan-induced rat paw edema assay (18), showed that this group of arylidene-2phenoxybenzoic acid hydrazides exhibit the reduction of rat paw edema (17-58% reduction in comparison with the control group and a moderate to good activity range in comparison with diclofenac as the reference drug (Table 2). As shown in Table 2, the presence of electronwithdrawing substituents (9d-e) produced compounds with comparable activity to unsubstituted derivative (9a), while electrondonating (9b-c) substituted compounds (9bc) showed to be less active in reduction of compounds showed better activity in 1 or 2 h rather than 3 h showing the faster onset of action compared to diclofenac which was possibly resulted from their more lipophilic structure. Poly-substitution in 9f-g resulted to a noticeable reduction in activity. Finally, N-(4-chlorobenzylidene)-2-(2-phenoxyphenyl) acetohydrazide (9d), was the most potent antithe administration of the drug.

Conclusion
Various substituted arylidene-2phenoxynicotinic acid hydrazide derivatives were synthesized and screened for their potential activities in comparison with diclofenac. Compounds 9a, 9d and 9e exhibited the most activity.